This project aims to define the roles of inositol signaling reactions in the pathogenesis of disease. We will study the role of inositol 1,3,4-5/6-kinase in fat cell differentiation. This enzyme catalyzes the first step in formation of inositol hexakisphosphate (IP6) which is essential for life in mammals. Preliminary studies indicate that the enzyme and others leading to IP6 production are highly expressed during fat cell differentiation and that some metabolite in this pathway may actually cause fat cell development. We will use overexpression and RNAi studies to define the causal molecule. In another study we plan to determine the potentially prothrombotic phenotype of mice with elevated levels of PI(3,4)P2. We propose that the elevated levels arise from deficiency of 4-ptaseI that occur in Weeble mutant mice. We have made radiation chimeric mice from lethally irradiated normal mice rescued by fetal liver transplants from Weeble embryos. Thus the Weeble mutation is restricted to blood cells and preliminary experiments suggest that these animals have a thrombotic phenotype. We will study platelet function and in vivo thrombosis in a laser injury model. We will investigate the functions of a little studied sub branch of the myotubularin PI 3-ptase family namely MTMR6, MTMR7, MTMR8 and their inactive partner MTMR9. These proteins play undefined roles in stress-induced apoptosis. We recently learned that mutations in another enzyme discovered in our lab are the cause of a severe neurodegeneration Jobert syndrome. The enzyme is inositol polyphosphate 5-phosphataseIV a lipid specific 5-PtaseIV. Dr Jos Gleeson (UCSD/HHMI) has found 5 different mutations in families with Joubert syndrome and has sent us cells and constructs to define the biological consequences of these mutations. In summary we use inositol biochemistry to determine the phenotype vs genotype of disorders of inositol metabolism.